Nucleic acid hybridization assays comprise an important class of techniques in modern biology, with diverse applications in diagnosis of inherited disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing. Primer extension reactions are key components of many nucleic acid hybridization assays and amplification methods. The polymerase chain reaction (PCR) amplification method has enabled advances in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like (Gilliland, 1990; Bevan, 1992; Green, 1991; McPherson, 1995).
The specificity and affinity of two or more strands of nucleic acid and nucleic acid analog molecules hybridizing by Watson/Crick and non-Watson/Crick base pairing are key parameters of efficient nucleic acid hybridization assays. Covalently attached labels, or moieties, which improve or stabilize hybridization are desirable.
Another important aspect of nucleic acid hybridization assays is the method used to facilitate detection of the hybridization event. Fluorescent methods have many advantages over radioisotopes where either the target polynucleotide or the probe or primer can be easily and safely labelled with fluorescent dyes. Methods to lower the costs of labelled probes and primers, or their functional equivalents, are highly desirable. There remains a need for further improvements in the specificity, affinity, and detection of nucleic acid hybridization assays.